A designer molecular chaperone against transmissible spongiform encephalopathy slows disease progression in mice and macaques

Abstract

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that lack therapeutic solutions. Here, we show that the molecular chaperone (N,N′-([cyclohexylmethylene]di-4,1-phenylene)bis(2-[1-pyrrolidinyl]acetamide)), designed via docking simulations, molecular dynamics simulations and quantum chemical calculations, slows down the progress of TSEs. In vitro, the designer molecular chaperone stabilizes the normal cellular prion protein, eradicates prions in infected cells, prevents the formation of drug-resistant strains and directly inhibits the interaction between prions and abnormal aggregates, as shown via real-time quaking-induced conversion and in vitro conversion NMR. Weekly intraperitoneal injection of the chaperone in prion-infected mice prolonged their survival, and weekly intravenous administration of the compound in macaques infected with bovine TSE slowed down the development of neurological and psychological symptoms and reduced the concentration of disease-associated biomarkers in the animals’ cerebrospinal fluid. The de novo rational design of chaperone compounds could lead to therapeutics that can bind to different prion protein strains to ameliorate the pathology of TSEs.

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Fig. 1: Logical design of MC and its anti-prion activity.
Fig. 2: Inhibition of the RT-QuIC process.
Fig. 3: IVC-NMR and the detection of TICs.
Fig. 4: Anti-prion effects of MC.
Fig. 5: Treatment of prion-infected mice with MC.
Fig. 6: Treatment of BSE-infected macaques with MC.
Fig. 7: Digital pathological analyses of BSE-infected macaques with or without MC treatment.

Code availability

The PAICS codes are available at www.paics.net/index_e.html.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information.

References

  1. 1.

    Prusiner, S. B. Novel proteinaceous infectious particles cause scrapie. Science 216, 136–144 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Prusiner, S. B. Prions. Proc. Natl Acad. Sci. USA 95, 13363–13383 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Safar, J. et al. Eight prion strains have PrPSc molecules with different conformations. Nat. Med. 4, 1157–1165 (1998).

    CAS  Article  Google Scholar 

  4. 4.

    Huang, Y., Gregori, L., Anderson, S. A., Asher, D. M. & Yang, H. Development of dose–response models of Creutzfeldt–Jakob disease infection in nonhuman primates for assessing the risk of transfusion-transmitted variant Creutzfeldt–Jakob disease. J. Virol. 88, 13732–13736 (2014).

    Article  Google Scholar 

  5. 5.

    Vazquez-Fernandez, E. et al. The structural architecture of an infectious mammalian prion using electron cryomicroscopy. PLoS Pathog. 12, e1005835 (2016).

    Article  Google Scholar 

  6. 6.

    Groveman, B. R. et al. Parallel in-register intermolecular beta-sheet architectures for prion-seeded prion protein (PrP) amyloids. J. Biol. Chem. 289, 24129–24142 (2014).

    CAS  Article  Google Scholar 

  7. 7.

    Govaerts, C., Wille, H., Prusiner, S. B. & Cohen, F. E. Evidence for assembly of prions with left-handed beta-helices into trimers. Proc. Natl Acad. Sci. USA 101, 8342–8347 (2004).

    CAS  Article  Google Scholar 

  8. 8.

    Anfinsen, C. B. Principles that govern the folding of protein chains. Science 181, 223–230 (1973).

    CAS  Article  Google Scholar 

  9. 9.

    Kabir, A. et al. Effects of ligand binding on the stability of aldo-keto reductases: implications for stabilizer or destabilizer chaperones. Protein Sci. 25, 2132–2141 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Uchiyama, K. et al. Prions amplify through degradation of the VPS10P sorting receptor sortilin. PLoS Pathog. 13, e1006470 (2017).

    Article  Google Scholar 

  11. 11.

    Berry, D. B. et al. Drug resistance confounding prion therapeutics. Proc. Natl Acad. Sci. USA 110, E4160–E4169 (2013).

    CAS  Article  Google Scholar 

  12. 12.

    Giles, K. et al. Optimization of aryl amides that extend survival in prion-infected mice. J. Pharmacol. Exp. Ther. 358, 537–547 (2016).

    CAS  Article  Google Scholar 

  13. 13.

    Ma, B., Yamaguchi, K., Fukuoka, M. & Kuwata, K. Logical design of anti-prion agents using NAGARA. Biochem. Biophys. Res. Commun. 469, 930–935 (2016).

    CAS  Article  Google Scholar 

  14. 14.

    Kuwata, K. Logical design of medical chaperone for prion diseases. Curr. Top. Med. Chem. 13, 2432–2440 (2013).

    CAS  Article  Google Scholar 

  15. 15.

    Kuwata, K. et al. Hot spots in prion protein for pathogenic conversion. Proc. Natl Acad. Sci. USA 104, 11921–11926 (2007).

    CAS  Article  Google Scholar 

  16. 16.

    Ishikawa, T., Ishikura, T. & Kuwata, K. Theoretical study of the prion protein based on the fragment molecular orbital method. J. Comput. Chem. 30, 2594–2601 (2009).

    CAS  Article  Google Scholar 

  17. 17.

    Milhavet, O. et al. Prion infection impairs the cellular response to oxidative stress. Proc. Natl Acad. Sci. USA 97, 13937–13942 (2000).

    CAS  Article  Google Scholar 

  18. 18.

    Kimura, T., Hosokawa-Muto, J., Kamatari, Y. O. & Kuwata, K. Synthesis of GN8 derivatives and evaluation of their antiprion activity in TSE-infected cells. Bioorg. Med. Chem. Lett. 21, 1502–1507 (2011).

    CAS  Article  Google Scholar 

  19. 19.

    Atarashi, R. et al. Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion. Nat. Med. 17, 175–178 (2011).

    CAS  Article  Google Scholar 

  20. 20.

    Saborio, G. P., Permanne, B. & Soto, C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411, 810–813 (2001).

    CAS  Article  Google Scholar 

  21. 21.

    Miller, M. B. et al. Cofactor molecules induce structural transformation during infectious prion formation. Structure 21, 2061–2068 (2013).

    CAS  Article  Google Scholar 

  22. 22.

    Riek, R. et al. NMR structure of the mouse prion protein domain PrP(121-231). Nature 382, 180–182 (1996).

    CAS  Article  Google Scholar 

  23. 23.

    Singh, J. & Udgaonkar, J. B. The pathogenic mutation T182A converts the prion protein into a molten globule-like conformation whose misfolding to oligomers but not to fibrils is drastically accelerated. Biochemistry 55, 459–469 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Yamamoto, N. & Kuwata, K. Regulating the conformation of prion protein through ligand binding. J. Phys. Chem. B 113, 12853–12856 (2009).

    CAS  Article  Google Scholar 

  25. 25.

    Singh, J. & Udgaonkar, J. B. Structural effects of multiple pathogenic mutations suggest a model for the initiation of misfolding of the prion protein. Angew. Chem. Int. Ed. Engl. 54, 7529–7533 (2015).

    CAS  Article  Google Scholar 

  26. 26.

    Honda, R. P., Xu, M., Yamaguchi, K., Roder, H. & Kuwata, K. A native-like intermediate serves as a branching point between the folding and aggregation pathways of the mouse prion protein. Structure 23, 1735–1742 (2015).

    CAS  Article  Google Scholar 

  27. 27.

    Sugase, K., Dyson, H. J. & Wright, P. E. Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021–1025 (2007).

    CAS  Article  Google Scholar 

  28. 28.

    Malevanets, A. et al. Interplay of buried histidine protonation and protein stability in prion misfolding. Sci. Rep. 7, 882 (2017).

    Article  Google Scholar 

  29. 29.

    Kamatari, Y. O., Hayano, Y., Yamaguchi, K., Hosokawa-Muto, J. & Kuwata, K. Characterizing antiprion compounds based on their binding properties to prion proteins: implications as medical chaperones. Protein Sci. 22, 22–34 (2013).

    CAS  Article  Google Scholar 

  30. 30.

    Gatti, J. L. et al. Prion protein is secreted in soluble forms in the epididymal fluid and proteolytically processed and transported in seminal plasma. Biol. Reprod. 67, 393–400 (2002).

    CAS  Article  Google Scholar 

  31. 31.

    Peralta, O. A. & Eyestone, W. H. Quantitative and qualitative analysis of cellular prion protein (PrPC) expression in bovine somatic tissues. Prion 3, 161–170 (2009).

    Article  Google Scholar 

  32. 32.

    Ono, F. et al. Experimental transmission of bovine spongiform encephalopathy (BSE) to cynomolgus macaques, a non-human primate. Jpn. J. Infect. Dis. 64, 50–54 (2011).

    CAS  PubMed  Google Scholar 

  33. 33.

    Orru, C. D. et al. Rapid and sensitive RT-QuIC detection of human Creutzfeldt–Jakob disease using cerebrospinal fluid. mBio 6, e02451-14 (2015).

    Article  Google Scholar 

  34. 34.

    International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) adopts consolidated guideline on good clinical practice in the conduct of clinical trials on medicinal products for human use. Int. Dig. Health Legis. 48, 231–234 (1997).

  35. 35.

    Mead, S. et al. PRION-1 scales analysis supports use of functional outcome measures in prion disease. Neurology 77, 1674–1683 (2011).

    CAS  Article  Google Scholar 

  36. 36.

    Matsui, Y. et al. High sensitivity of an ELISA kit for detection of the gamma-isoform of 14-3-3 proteins: usefulness in laboratory diagnosis of human prion disease. BMC. Neurol. 11, 120 (2011).

    CAS  Article  Google Scholar 

  37. 37.

    Kawasaki, Y. et al. Orally administered amyloidophilic compound is effective in prolonging the incubation periods of animals cerebrally infected with prion diseases in a prion strain-dependent manner. J. Virol. 81, 12889–12898 (2007).

    CAS  Article  Google Scholar 

  38. 38.

    Lysek, D. A. et al. Prion protein NMR structures of cats, dogs, pigs, and sheep. Proc. Natl Acad. Sci. USA 102, 640–645 (2005).

    CAS  Article  Google Scholar 

  39. 39.

    Nicoll, A. J. et al. Pharmacological chaperone for the structured domain of human prion protein. Proc. Natl Acad. Sci. USA 107, 17610–17615 (2010).

    CAS  Article  Google Scholar 

  40. 40.

    Massignan, T. et al. A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein. Sci. Rep. 6, 23180 (2016).

    CAS  Article  Google Scholar 

  41. 41.

    Zahn, R. et al. NMR solution structure of the human prion protein. Proc. Natl Acad. Sci. USA 97, 145–150 (2000).

    CAS  Article  Google Scholar 

  42. 42.

    Gossert, A. D., Bonjour, S., Lysek, D. A., Fiorito, F. & Wuthrich, K. Prion protein NMR structures of elk and of mouse/elk hybrids. Proc. Natl Acad. Sci. USA 102, 646–650 (2005).

    CAS  Article  Google Scholar 

  43. 43.

    Garcia, F. L., Zahn, R., Riek, R. & Wuthrich, K. NMR structure of the bovine prion protein. Proc. Natl Acad. Sci. USA 97, 8334–8339 (2000).

    CAS  Article  Google Scholar 

  44. 44.

    Calzolai, L., Lysek, D. A., Perez, D. R., Guntert, P. & Wuthrich, K. Prion protein NMR structures of chickens, turtles, and frogs. Proc. Natl Acad. Sci. USA 102, 651–655 (2005).

    CAS  Article  Google Scholar 

  45. 45.

    Oelschlegel, A. M. & Weissmann, C. Acquisition of drug resistance and dependence by prions. PLoS Pathog. 9, e1003158 (2013).

    CAS  Article  Google Scholar 

  46. 46.

    Ghaemmaghami, S. et al. Continuous quinacrine treatment results in the formation of drug-resistant prions. PLoS Pathog. 5, e1000673 (2009).

    Article  Google Scholar 

  47. 47.

    Demaimay, R. et al. Late treatment with polyene antibiotics can prolong the survival time of scrapie-infected animals. J. Virol. 71, 9685–9689 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Lee, W., Tonelli, M. & Markley, J. L. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31, 1325–1327 (2015).

    Article  Google Scholar 

  49. 49.

    Delano, W. L. Use of PyMOL as a communications tool for molecular science. Abstr. Pap. Am. Chem. Soc. 228, U313–U314 (2004).

    Google Scholar 

  50. 50.

    Kitaura, K., Ikeo, E., Asada, T., Nakano, T. & Uebayasi, M. Fragment molecular orbital method: an approximate computational method for large molecules. Chem. Phys. Lett. 313, 701–706 (1999).

    CAS  Article  Google Scholar 

  51. 51.

    Ishikawa, T. & Kuwata, K. Fragment molecular orbital calculation using the RI-MP2 method. Chem. Phys. Lett. 474, 195–198 (2009).

    CAS  Article  Google Scholar 

  52. 52.

    Kay, L. E., Torchia, D. A. & Bax, A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28, 8972–8979 (1989).

    CAS  Article  Google Scholar 

  53. 53.

    Nishida, N. et al. Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein. J. Virol. 74, 320–325 (2000).

    CAS  Article  Google Scholar 

  54. 54.

    Hosokawa-Muto, J., Kamatari, Y. O., Nakamura, H. K. & Kuwata, K. Variety of antiprion compounds discovered through an in silico screen based on cellular-form prion protein structure: correlation between antiprion activity and binding affinity. Antimicrob. Agents Chemother. 53, 765–771 (2009).

    CAS  Article  Google Scholar 

  55. 55.

    Thompson, A. G. et al. The Medical Research Council prion disease rating scale: a new outcome measure for prion disease therapeutic trials developed and validated using systematic observational studies. Brain 136, 1116–1127 (2013).

    Article  Google Scholar 

  56. 56.

    Muramatsu, S. et al. Behavioral recovery in a primate model of Parkinson’s disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes. Hum. Gene Ther. 13, 345–354 (2002).

    CAS  Article  Google Scholar 

  57. 57.

    R Development Core Team R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2014).

  58. 58.

    Takahashi, H. et al. Characterization of antibodies raised against bovine-PrP-peptides. J. Neurovirol. 5, 300–307 (1999).

    CAS  Article  Google Scholar 

  59. 59.

    Meredith, J. E. Jr et al. Characterization of novel CSF Tau and ptau biomarkers for Alzheimer’s disease. PLoS ONE 8, e76523 (2013).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank M. Fukushima at the Translational Research Informatics Center and H. Mizusawa at the National Center of Neurology and Psychiatry for fruitful discussion. We also thank R. Honda, T. Saeki, M. Horii and S. Hori for providing technical help. K.K. was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by grants from the Ministry of Health, Labour and Welfare. The study was also supported by a grant from the Practical Research Project for Rare/Intractable Diseases of the Japan Agency for Medical Research and Development.

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Authors

Contributions

K.Y. prepared the 15N-labelled and non-labelled recombinant prions, and performed the IVC-NMR measurements and analysis. Y.O.K. measured and analysed the binding data using NMR and SPR. F.O. and H.S. conducted the in vivo treatment study. D.I. and M.T. conducted the pathological examinations for mice and macaques, respectively. T.F. and N.N. analysed Tau proteins in the central nervous system. T.K. established the method for the synthesis of the anti-prion compounds. J.H-M., A.E.E. and M.F. performed the in vitro, ex vivo and in vivo experiments. T.I. coded the FMO programme PAICS using the programming language C. Y.T. and Y.M. performed the statistical analysis. K.K. supervised the project, analysed the data and wrote the manuscript.

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Correspondence to Kazuo Kuwata.

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Supplementary Video 1

Prion infected macaque (number 6, control) at 19.7 m.p.i.

Supplementary Video 2

Prion infected macaque (number 3, BOS) at 19.7 m.p.i.

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Yamaguchi, K., Kamatari, Y.O., Ono, F. et al. A designer molecular chaperone against transmissible spongiform encephalopathy slows disease progression in mice and macaques. Nat Biomed Eng 3, 206–219 (2019). https://doi.org/10.1038/s41551-019-0349-8

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